Optical sensor and optical process for the characterization of a chemical and/or bio-chemical substance

ABSTRACT

The optical sensor contains an optical waveguide ( 1 ) with a substrate ( 104 ), waveguiding material ( 105 ), a cover medium ( 106 ) and a waveguide grating structure ( 101 - 103 ). By means of a light source ( 2 ), light can be emitted to the waveguide grating structure ( 101 - 103 ) from the substrate side and/or from the cover medium side. ( 101 - 103 ). With means of detection ( 11 ), at least two differing light proportions ( 7 - 10 ) radiated from the waveguide ( 1 ) can be detected. For carrying out a measurement, the waveguide can be immovably fixed relative to the light source ( 2 ) and the means of detection ( 11 ). The waveguide grating structure ( 101 - 103 ) itself consists of one or several waveguide grating structure units ( 101 - 103 ), which if so required can be equipped with (bio-)chemo-sensitive layers. The sensor permits the generation of absolute measuring signals.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. application Ser. No.09/508,384 filed Jun. 19, 2000, now U.S. Pat. No. 6,455,004, issued Sep.24, 2002, which application is hereby incorporated herein by reference.

BACKGROUND OF INVENTION

[0002] The invention concerns an optical sensor and an optical processfor the characterization of a chemical and/or bio-chemical substance.

[0003] Waveguide grating structures with and without a chemo-sensitivelayer are described in the literature (refer to, e.g., EP 0 226 604 B1,EP 0 482 377 A2, PCT WO 95/03538, SPIE Vol. 1141, 192-200, PCT WO97/09594, Advances in Biosensors 2 (1992), 261-289, U.S. Pat. No.5,479,260, SPIE Vol. 2836, 221-234).

[0004] In EP 0 226 604 B1 and EP 0 482 377 A2, it is demonstrated, howthe effective refractive index (resp., the coupling angle) of achemo-sensitive grating coupler can be measured as a sensor signal. Thesensor signal “effective refractive index” or “coupling angle” is avalue which manifests a strong dependence on temperature.

[0005] Front-side in-coupling of light into a waveguide (refer to SPIEVol. 1141, 192-200) is not practical, because a high positioningaccuracy is required. In addition, the front side of the wave guide hasto be of good optical quality. In PCT WO 95/03538 it is demonstrated howthe absolute out-coupling angle of a mode is measured. This value,however, without referencing manifests a high dependence on temperature.In PCT WO 97/09594, chirped waveguide gratings are presented, which,however, also manifest a dependence on temperature.

[0006] In Advances in Biosensors 2 (1992), 261-289 it is shown how thedisturbing “pore diffusion” can be referenced away with the three-layerwaveguide model. The refractive index of the waveguiding film manifestsdrift, while the layer thickness of the waveguiding film (=sensorsignal) remains stable. The layout is designed with movable mechanics,which does not permit rapid measurements. In addition, the sensor signalor the light emerging from the waveguide grating structure is recordedfrom the front side. Front side detection is unsuitable for atwo-dimensional layout of waveguide grating structure units.Furthermore, the effective refractive indexes N(TE) and N(TM) for thetwo polarizations TE and TM are not simultaneously recorded, because forthe recording of resonance coupling curves separated by angle amechanical angular scan is carried out.

[0007] In U.S. Pat. No. 5,479,260, a bi-diffractive or multi-diffractivegrating coupler is described, whereby the sensor signal is produced bythe interferometry of two out-coupling beams of the same or of differingpolarization (with the use of a polarizer). Interferometric measurementsare complicated, because the intensities of the two beams have to bematched to one another. In addition, temperature fluctuations due to theinterferometric signal generated by differing polarizations (using apolarizer) are only partially compensated.

[0008] In SPIE Vol. 2836, 221-234, a layout for a waveguide gratingstructure in connection with fluorescence or luminescence measurementsis described. This layout, however, is not suitable for an (if necessarysimultaneous) (absolute) temperature-compensated measurement on thebasis of a direct detection. In addition, the waveguide gratingstructure is mounted on a revolving table.

[0009] In Applied Optics 20 (1981), 2280-2283, a temperature-independentoptical waveguide is described, whereby the substrate is made ofsilicon. Silicon is absorbent in the visual spectral range. In the caseof chemo-sensitive waveguide grating structures, however, thein-coupling takes place in preference from the substrate side. Inaddition, Applied Optics 20 (1981), 2280-2283, grating couplers whichare not temperature-independent are described.

SUMMARY OF INVENTION

[0010] The invention presented here has the object of creating a(bio-)chemo-sensitive optical sensor and to indicate an optical processfor the characterization of a (bio-) chemical substance, which do nothave the above disadvantages. With the invention, in particular:

[0011] (1) sensor signals can be generated, which manifest a lowdependence on temperature and/or a low dependence on the diffusion ofthe specimen liquid into the micropores of a waveguiding film;

[0012] (2) both the measurement of (absolute) sensor signals withrespect to a direct detection (absolute out-coupling angles α(TE) andα(TM) for the TE- or TM-wave, effective refractive indexes N(TE) andN(TM) for the TE- or TM-wave, layer thickness t_(F) of the waveguidingfilm etc.) as well as the measurement of (absolute) sensor signals withrespect to a marking detection (referenced fluorescence-, luminescence-,phosphorescence signals, etc.), are possible; and

[0013] (3) sensor signals remain stable with respect to a slight tiltingand/or displacement of the waveguide grating structure, because (localand angular) differences of sensor signals or referenced sensor signalsare measured.

[0014] The optical sensor according to the invention contains at leastone optical waveguide with a substrate, waveguiding material, a covermedium and at least one waveguide grating structure, at least one lightsource, by means of which light can be emitted from the substrate sideand/or from the cover medium side onto at least a part of the waveguidegrating structure, and means for the detection of at least two differinglight proportions, whereby with at least one detection agent lightemitted into the substrate and/or cover medium can be detected, wherebyfor the carrying out of a measurement the waveguide can be fixedimmovably with respect to the at least one light source and the means ofdetection.

[0015] In the case of the optical process according to the invention forthe characterization of a chemical and/or bio-chemical substance in aspecimen by means of an optical waveguide containing at least onewaveguide grating structure, the specimen is brought into contact withthe waveguide in at least one contact zone, in the waveguide in theregion of the at least one contact zone at least one light wave isexcited through the waveguide grating structure, the at least one lightwave is brought into interaction with the specimen, light in at leasttwo differing proportions is detected, of which at least one proportionoriginates from the region of the contact zone, and at least oneabsolute measuring signal is generated by the evaluation of the detectedlight.

[0016] The waveguide grating structure consists of one or severalwaveguide grating structure units, which are arranged one-dimensionallyor two-dimensionally (e.g., in a matrix shape or circular shape).

[0017] A possible xy displacement (or only an x-displacement) of thereading head (the reading heads) from one waveguide grating structure tothe other or a possible xy-displacement (or only x-displacement) of thewaveguide grating structure can quite well be applied.

[0018] A waveguide grating structure unit consists of at least two“sensing pads” (sensor platforms, sensor paths), which differ from oneanother in at least one of the characteristics:

[0019] (a) The light waves guided in the “sensing pads” differ in theirpolarization (TE-wave or TM-wave), whereby the generated sensor signalis not produced by interferometric measurement.

[0020] (b) The light waves guided in the “sensing pads” differ in theirmode number.

[0021] (c) The two chemo-sensitive layers assigned to the “sensing pads”manifest a differing specificity (ligand 1 selectively binds (inside oron the surface) to the chemo-sensitive layer covering the “sensing pad1”; ligand 2 selectively binds (inside or on the surface) to thechemo-sensitive layer covering the “sensing pad 2”).

[0022] (d) The chemo-sensitive layer assigned to the first “sensing pad”manifests specificity for one ligand (with or without “non-specificbinding”), while the (chemo-sensitive) layer assigned to the second“sensing pad” manifests no specificity (with or without “non-specificbinding”) (example: Dextran layer, to which no identification molecule(e.g., an antibody) is bound).

[0023] (e) The light waves guided in the “sensing pads” differ in theirwavelength.

[0024] A “sensing pad”, in which guided light waves of differingpolriztion (TEwave or TM-wave) are excited, counts as two “sensing pads”(difference in the polarization!), providing the sensor signal generatedis not produced by interferometric measurement.

[0025] A “sensing pad”, in which guided light waves of differing modenumber are excited, counts as two “sensing pads” (difference in the modenumber!).

[0026] A “sensing pad”, in which guided light waves of differingwavelengths are excited, counts as two “sensing pads” (difference in thewavelength!).

[0027] The first and second “sensing pad” can also be considered assignal and reference path. The two “sensing pads” can (but do not haveto) have the same structure.

[0028] The (bio-)chemo-sensitive contacts the waveguiding film in acontact zone. This contact zone normally in the case of the directdetection contains at least one grating. (In the case of interferometricmeasurements with the same polarization, for a direct detection the(bio-)chemo-sensitive layer can also only be located between twogratings (also refer to EP 0 226 604)). (In the case of interferometricmeasurements with two differing polarizations (using a polarizer), the(bio-)chemo-sensitive layer can also be located on a multi-diffractive(bi-diffractive) grating (refer to U.S. Pat. No. 5,479,260)).

[0029] On principle, for example, the value S(signal path) cS(referencepath) can serve as a possible referenced sensor signal, whereby S(signalpath) and S(reference path) are the sensor signals in the first “sensingpad” (signal path) or in the second “sensing pad” (reference path) and cis a calibration factor. In the case of the same polarization, sensiblyc=1. In the case of different polarizations, with c the differingsensitivities of the two polarizations can be taken into account. In thecase of different wavelengths or mode numbers, with c the differingsensitivities of the wavelengths or of the mode numbers can be takeninto account. Advantageously, signal path and reference path are asclose together as possible.

[0030] The referenced sensor signal in the case of the same polarizationfurthermore has the advantage that disturbances δ, such as, e.g., thosecaused by temperature fluctuation, light wavelength fluctuation,undesired diffusion of molecules in the waveguides, resp. in thechemo-sensitive layer, unspecific bindings, fluctuations of theconcentration of the molecules not to be detected, etc., or combinationsof these, can be referenced away, i.e., the referenced sensor signal isindependent of δ, because S(signal path)+δ−(S(referencepath)+δ)=S(signal path)−S(reference path).

[0031] In preference, mono-mode waveguides are utilized, which onlycarry the fundamental TE-mode or only the fundamental TE-mode and thefundamental TM-mode. The waveguiding film should preferably consist of ahigh-refractive material, which guarantees the generation of highsensitivities. The waveguiding film can be coated with a chemo-sensitivelayer (e.g., an anti-body layer (e.g., suitable for the detection of acorresponding antigen), a dextran layer with identification molecule(e.g., anti-bodies), receptors, DNA-sections, a silicon layer for thedetection of hydrocarbons, etc.). The waveguiding film itself, however,can also represent a chemo-sensitive layer. Rib waveguides can also beutilized.

[0032] A “sensing pad” comprises at least one grating, but can alsocomprise a (possibly more strongly modulated) in-coupling grating and atleast one out-coupling grating.

[0033] The grating periods of in-coupling grating and out-couplinggrating can be different (and are in most cases different).

[0034] In-coupling grating and out-coupling grating can beuni-diffractive or multi-diffractive grating structures (bi-diffractivegratings, gratings with changing grating period and/or with changinggrating diffraction vector, etc.).

[0035] A preferred “sensing pad” arrangement consists of three gratings,whereby the middle grating represents the in-coupling grating and thetwo outer gratings two out-coupling gratings. With a strongly modulatedin-coupling grating, for example, one succeeds in exciting modes inforward and reverse direction with a sole (resp., with two) incident (ifso required slightly focussed) light beam(s). While a slightdisplacement of the incident light beam in the mode propagationdirection or a slight tilting of the waveguide grating structure withrespect to the incident light beam (in the plane of incidence) changethe intensity of the modes (running in forward and/or reverse direction)as a result of the changed coupling geometry, not, however, theout-coupling angle(s) or the difference of the out-coupling angles(=double absolute out-coupling angle), which represent possible sensorsignals.

BRIEF DESCRIPTION OF DRAWINGS

[0036]FIG. 1 shows the above preferred “sensing pad” arrangement bothfor the TE-mode as well as for the TM-mode, whereby the two “sensingpads” are located adjacent to one another.

[0037]FIG. 2 illustrates further waveguide grating structure units.

[0038]FIG. 3 shows an optical sensor according to the invention.

DETAILED DESCRIPTION

[0039] A further preferred “sensing pad” arrangement also consists ofthree gratings, whereby the two outer gratings form (possibly morestrongly modulated) in-coupling gratings and the middle grating theout-coupling grating.

[0040] In case of the detection of (bio-)molecular interactions with thearrangement according to FIG. 1 (or equivalent arrangements), thewaveguide structure unit is coated with a (bio-)chemo-sensitive layer,to which then in the experiment a specific binding partner binds, whichleads to a change of the out-coupling angles α(TE)=(α(TE+)−α(TE-))/2 andα(TM)=(α(TM+)−α(TM−))/2 (notation: e.g., α(TE+): out-coupling angle ofthe TEmode running in +xdirection or to a change of the effectiverefractive indexes N(TE) and N(TM) and of the integrated optical valuesderivable from it such as, e.g., of the layer thickness t_(F) of thewaveguiding film in the three-layer waveguide model (see further below).Different waveguide grating structure units can be coated with differentchemo-sensitive layers.

[0041] The out-coupling angle of the two out-coupling gratings (resp.,of the out-coupling grating) of the preferred “sensing pad” arrangementenable an absolute determination of the out-coupling angle (resp., ofthe effective refractive index), although the out-coupling of the twolight beams does not take place at one and the same location.

[0042] In case of two adjacent “sensing pads” for the TE- and theTM-mode according to the preferred “sensing pad” arrangement (see FIG.1), the out-coupling even takes place at four different locations.

[0043] The “sensing pad” for the TE-mode enables the determination ofthe effective refractive index N(TE) of the TE-mode. The “sensing pad”for the TM-mode enables the determination of the effective refractiveindex N(TM) of the TM-mode. In case of simultaneous illumination of thetwo “sensing pads” (with, e.g., a single light field with 45° linearpolarized light or circular (or elliptical) polarized light), thedetermination of the out-coupling angle (of the effective refractiveindex) for the TE- and TM-mode can take place simultaneously.

[0044]FIG. 1 illustrates an advantageous embodiment of a waveguidegrating structure unit. The “sensing pad” for the TE-mode consists of anin-coupling grating G_(i) (TE) and the two out-coupling gratings G_(o+)(TE) und G_(o−) (TE) located on the left and right of the in-couplinggrating. The sensing pad for the TM-mode consists of the in-couplinggrating G_(i) (TM) and the two out-coupling gratings G_(o+) (TM) andG_(o−) (TM) located on the left and right of the in-coupling grating andit is located adjacent to the “sensing pad” for the TE-mode. In order tokeep the influence of disturbances (e.g., temperature fluctuations) low,the two “sensing pads” should be located as closely as possible adjacentto one another. The grating lines are aligned parallel to the yaxis.

[0045] It is advantageous if the out-coupling gratings G_(o+) (TE),G_(o−) (TE), G_(o+) (TM), G_(o−) (TM) are completely illuminated by theguided light waves, as a result of which it is ensured that in case ofthe respective out-coupling grating the sensor surface corresponds tothe surface of the out-coupling grating. This is achieved by selecting(a) the width of the out-coupling grating in y-direction smallerthan/equal to the width of the in-coupling grating in y-direction and(b) the illumination spot on the in-coupling grating having a y-width,which is greater than/equal to the y-width of the out-coupling gratings,and the illumination spot including the complete y-width of anout-coupling grating.

[0046] An advantageous embodiment is the one, in the case of which thetwo in-coupling gratings G_(i) (TE) and G_(i) (TM) are simultaneouslyilluminated with one (or two (wedge-shaped) light field(s) linearpolarized under 45°. With this, simultaneously the TE-mode and theTM-mode (in the forward and reverse direction) can be excited andsimultaneously all (in the case presented here: four) out-couplingangles can be measured. G_(i) (TE) and G_(i) (TM) in preference havediffering grating periods.

[0047] It is, however, also possible that a “sensing pad” as suchcarries out the function of a “sensing pad” for the TE-mode and thefunction of a “sensing pad” for the TM-mode. The second “sensing pad”(located adjacent to it) is then either not present or else is utilizedas a check or reference. (This reference “sensing pad” can, e.g., becoated with a second chemo-sensitive layer or with a non-specific layer,i.e., a layer which has no specificity (with or without “non-specificbinding”). If the in-coupling grating of a “sensing pad” is illuminatedwith TE-light as well as with TM-light under the correspondingin-coupling angles (e.g., with 45° linear polarized light), then in theone “sensing pad” both the TE-mode as well as the TM-mode are excited(if so required both in forward as well as in reverse direction).

[0048] It is also possible to excite the TE-mode only in forwarddirection and the TM-mode only in reverse direction (or vice-versa) orthe TE-mode and the TM-mode only in forward direction (or vice-versa).In such a case, then the corresponding two out-coupling angles or theirdifference are measured.

[0049] Utilized as detectors are advantageously one- or two-dimensional(digital or analogue) position-sensitive detectors (PSD), photo-diodearrays, CCD line- or surface camera(s), etc. The out-coupled light beamsare advantageously focussed with a lens, whereby the focus does notindispensably have to be situated exactly on the detector surface. Theout-coupling gratings can also be focusing gratings. This has thebenefit that the lens-effect is already integrated in the out-couplinggrating.

[0050] It is also possible to select the direction of the grating linesof the out-coupling grating in such a manner, that the grating lines arevertical to the propagation direction of the mode. Advantageously, thetilting of the grating lines (resp., of the grating diffraction vectors)of the out-coupling gratings of two adjacent “sensing pads” has aninverse operational sign. With this, a better separation of theout-coupled light waves can be achieved.

[0051] The in-coupling grating can also be selected in such a manner,that the grating period in xdirection or ydirection does not remainconstant or is graduated. The requirements of the accuracy of thesetting of the angle of incidence are relaxed as a result of this.

[0052] The in-coupling gratings shown in FIG. 1 in preference have astrong modulation and are characterized by a short width in x-direction.As a result, the requirements of the accuracy of the setting of theangle of incidence are relaxed, because the light can now in-couple froma greater angle segment.

[0053] FIGS. 2(a) and 2(b) illustrate two further embodiments ofwaveguide grating structures, whereby here the dimension of thein-coupling gratings in ydirection is smaller than that of theout-coupling gratings. In FIG. 2(b), a uniform out-coupling gratingG_(O−) for the TE- and TM-wave running in (−x) direction and a uniformout-coupling grating G_(O+) for the TE- and TM-wave running in (+x)direction are depicted.

[0054]FIG. 3 illustrates an embodiment of a sensor according to theinvention. The sensor contains a waveguide 1 with a substrate 104, awaveguiding material 105 and a cover medium 106 as well as with awaveguide grating structure according to FIG. 1.

[0055] A light source 2 generates, for example, 45° linearly polarizedlight; for this purpose, a linearly polarized laser with a polarizationplane inclined to the drawing plane by 45° can be utilized. Through abeam splitter 3 or a mirror 4, two impinging light beams 5, 6 areproduced. The light beams 5, 6 impinge on the two in-coupling gratings101 (of which in the side view of FIG. 3 only one is visible) of the two“sensing pads” through the substrate 104 and in the first “sensing pad”generate a TMwave in forward and reverse direction. The light waves areout-coupled through four out-coupling gratings 102, 103 (of which in theside view of FIG. 3 only two are visible) from the waveguide gratingstructure and radiated into the substrate 104. After passing through thesubstrate 104 they spread as light fields 7-10 and impinge on atwo-dimensional CCD-detector 11. If so required, there can be a lenssystem (not illustrated) between the waveguide 1 and the detector 11.

[0056] If in the two “sensing pads” one operates with differingpolarization and with a chemo-sensitive layer assigned to the waveguidegrating structure, then, for example, the sensor signal S=t_(F)(=layerthickness of the waveguiding film in the three-layer waveguide model)can be generated (also see further below). If now a second waveguidegrating structure unit, which also permits the generation of the sensorsignal S=t_(F), is laid next to the first waveguide grating structureunit (now to be considered as a whole as signal path) and the secondwaveguide grating structure unit (now to be considered as a whole asreference path) coated with, for example, a chemo-sensitive layer ofdiffering specificity or with a non-specific chemo-sensitive layer withor without “non-specific binding” (e.g., a dextran layer, to which noidentification molecule is bound), then, for example, the value t_(F)(measured in the signal path)−t_(F) (measured in the reference path) canserve as sensor signal.

[0057] Athough the effective refractive indexes N(TE) and N(TM) of theTE- or TM-mode are possibly not measured at the same point (the two“sensing pads” are, however, in the case of a chemo-sensitive sensorcoated with the same chemo-sensitive layer), with the waveguide gratingstructure unit the interesting sensor signals S and/or ΔS with

ΔS=Δα(TM)−Δα(TE)=Δ(α(TM)−α(TE))(ΔS=Δ(α(TE)−α(TM)))

(α=out-coupling angle)

ΔS=ΔN(TM)−ΔN(TE)(ΔS=ΔN(TE)−ΔN(TM)),

[0058] whereby the layer thickness of the waveguiding film in preferenceshould be selected in such a manner, that for the sensitivity of therefractive index of the cover medium ∂N(TE)/∂n_(C)=∂N(TM)/∂n_(C) isapplicable, which at least eliminates the influence the refractive-indexchanges of the cover medium on the sensor signal have on the sensorsignal (the refractive index n_(C) of the cover medium depends on thetemperature, i.e., n_(C)=n_(C)(T), whereby T is the temperature),

ΔS=Δt _(F)=const ((∂N(TE)/∂T)⁻¹ ΔN(TE)−(∂N(TM)/∂T)⁻¹ ΔN(TM))

[0059] wherebyconst=((∂N(TE)/∂T)⁻¹(∂N(TE)/∂t_(F))−(∂N(TM)/∂T)⁻¹(∂N(TM)/∂t_(F)))⁻¹ andthe change of the layer thickness Δt_(F) is calculated from the systemof equations

ΔN(TE)=(∂N(TE)/∂t _(F))Δt _(F)+(∂N(TE)/∂T)ΔT  (equation 1)

ΔN(TM)=(∂N(TM)/∂t_(F))Δt _(F)+(∂N(TM)/∂T)ΔT

[0060] and ∂N/∂T is the temperature coefficient of the waveguidecomplete with cover (=specimen) for the corresponding mode (∂N/∂T iscalculated taking into account the temperature coefficient (dN/dT)GRATING of the grating experimentally (or according to theory) (also seefurther below)),

[0061] ΔS=Δt_(F), whereby the layer thickness t_(F) is calculated withthe three-layer waveguide model (with N(TE) and/or N(TM) as inputparameter),

[0062] ΔS=Δt_(A) whereby the change of the additional layer thicknessA_(t) is calculated from the system of equations

ΔN(TE)=(∂N(TE)/∂t _(A))Δt _(A)+(∂N(TE)/∂T)ΔT  (equation 2)

ΔN(TM)=(∂N(TM)/∂t _(A))Δt _(A)+(∂N(TM)/∂T)ΔT,

[0063] ΔS=Δt_(A), whereby the additional layer thickness t_(A) iscalculated with the four-layer waveguide model (resp., five-layerwaveguide model),

[0064] ΔS=ΔΓ, whereby the mass density by surface Γ (refer to Chem.Commun. 1997, 1683-1684) is calculated with the four-layer waveguidemodel (or also with the three-layer waveguide model with the utilizationof approximations (refer to J. Opt. Soc. Am. B, Vol. 6, No.2, 209-220),etc., are measured. The above sensor signals are interesting, becausethey manifest a low dependence on temperature. On principle, however,during the measurement it still has to be taken into account that alsothe grating period manifests a temperature-dependence as a result of thethermal expansion coefficient of the sensor chip. Applicable is ΔΛ=αΛΔT,whereby ΔΛ is the change of the grating period Λ, ΔT the change of thetemperature T and α the thermal expansion coefficient (typically 4.5 10K⁻¹ for glass and 6.1 10 K⁻¹ for polycarbonate). The(dN/dT)_(GRATING)=I(λ/Λ) α caused by the grating, whereby I is the orderof diffraction and λ the wavelength, contributes to the temperaturecoefficient (dN/dT)_(CHIP) (=experimental measured value) of thecomplete sensor chip as follows:(dN/dT)_(CHIP)=(dN/dT)_(WAVEGUIDE+SPECIMEN)+(dN/dT)_(GRATING), whereby(dN/dT)_(WAVEGUIDE+SPECIMEN) is the temperature coefficient of thewaveguide complete with cover (=specimen).

[0065] The sensor signals S and ΔS can be recorded in function of thetime. One can, however, also only measure and compare an initial and afinal condition on a waveguide grating structure unit, whereby, forexample, in the interim other waveguide grating structure units can beevaluated or the even the waveguide grating structure can be removedfrom the measuring unit in the meantime, because absolute angles ordifferences of angles (resp., distances (differences of distances) oflight points) are measured and these values remain stable with respectto a tilting or displacement. The determination of a measured value canbe carried out by means of an individual measurement, also, however, bymeans of a statistical evaluation (e.g., averaging) of severalindividual measurements.

[0066] If apart from temperature changes ΔT, other interferences arealso present, such as, e.g., diffusion of the specimen liquid into themicro-pores of a porous waveguiding film (which leads to a change in therefraction index Δn_(F) of the waveguiding film) or tensions (such as,e.g., compressive strain or tensile stress) or changes in stress arepresent or if in general only pore diffusion is present, then (in thefirst case under certain conditions (e.g., the course in function oftime of the pore diffusion with the temperature T as group of curvesparameter is known) (approximately), then a (common) interference valueξ with N(t_(F), ξ)=N(t_(F), n_(F) (ξ), n_(S)(ξ), n_(C)(ξ)) or N(t_(A),ξ)=N(t_(F), t_(A), n_(A)(ξ)n_(F)(ξ), n_(S)(ξ), n_(C)(ξ)) can beintroduced. Equation (1) or (2) then take on the form

ΔN(TE)=(∂N(TE)/∂S)ΔS+(∂N(TE)/∂ξ)Δξ  (equation 3)

ΔN(TM)=(∂N(TM)/∂S)ΔS+(∂N(TM)/∂ξ)Δξ,

[0067] whereby ΔS is the sensor signal ΔT_(F) (or Δt_(A)) and, forexample, in the case of the three-layer waveguide model∂N/δξ=(∂N/∂n_(F))(∂n_(F)/∂ξ)+(∂N/∂n_(S))(∂n_(S)/∂ξ)+(∂N/∂n_(C))(∂n_(C)/∂ξ)(+(∂N/∂t_(F))(∂t_(F)/∂ξ),providing t_(F) is=t_(F)(ξ)). Logically directly from ξ=T formula (1) orformula (2) follows. ξ=n_(F), describes, e.g., the diffusion at aconstant temperature.

[0068] Also in the case of the presence of several interferences, it ispossible to operate with the three-layer waveguide model and the modesequation solved (example: input: N(TE), N(TM), output: t_(F), n_(F)(=refractive index of the waveguiding film) or t_(F), n_(C) (=refractiveindex of the cover medium) or t_(F), n_(S) (=refractive index of thesubstrate)). In the three-layer waveguide model, waveguiding film,chemo-sensitive layer and specific binding partner are considered asbeing a layer with one refractive index. Although n_(F) and n_(C) (andif applicable n_(S)) are not precisely known and if applicable changeduring the experiment on the basis of an interference, the measuredvalue t_(F) is relatively independent of the interference, dependent onthe contrary, however, on the binding process. The interference isso-to-say packed into the second output value, the parameter “refractiveindex”.

[0069] Fundamentally, pore diffusion and temperature fluctuations areindependent manifestations and should be described by two interferencevalues ξ₁ and ξ₂ (generalization: k independent disturbances aredescribed by k independent disturbance values ξ₁, . . . , ξ_(k)). FromN(t_(F), ξ₁, . . . , ξ_(k)) for both polarizations, for a given modenumber and for a given wavelength follows

ΔN=(∂N/∂t _(F))Δt_(F)+(∂N/∂ξ ₁)Δξ₁+ . . . +(∂N/∂ξ _(k))Δξ_(k).  (4)

[0070] Because the disturbance values can influence the waveguideparameters t_(F), n_(F), n_(S), n_(C), the following is applicable:

∂N/δξ _(i)=(∂N/δt _(F))(∂t _(F)/∂ξ_(i))+(∂N/n _(F))(∂n_(F)/∂ξ_(i))+(∂N/∂n _(S))(∂n_(S)/∂ξ_(i))+∂N/∂n _(C))(∂n_(C)/∂ξ_(i))  (equation 5)

[0071] (1<=i<=k) (the analogue is also applicable for N(t_(A), ξ₁, . . ., ξ_(k))). For the pore diffusion (ξ=n_(F)) and the temperature change(ξ₂=T) from Eq. (4) results

ΔN=(∂N/∂t _(F))Δt_(F)+(∂N/∂n _(F))Δn _(F)+(∂N/∂T)ΔT  (equation 6)

[0072] for both polarizations, a given mode number and a givenwavelength. ∂N/∂T can, e.g., be determined experimentally. If, e.g.,both polarizations are measured, then (6) represents two equations withthree unknown values. If, however, measurements are carried out atseveral wavelengths (taking into account the refractive indexdispersions) and/or mode numbers, then several systems of equationsrespectively consisting of three equations and three unknown values canbe put together and solved. If the dispersion in Δn_(F) (λ) is takeninto account as an unknown value (λ=wavelength), then one obtains, e.g.,with two wavelengths λ₁ and λ₂ and two polarizations four equations withfour unknown values Δt_(F), Δn_(F) (λ₁), Δn_(F) (λ), ΔT and from themdetermines the unknown values. The value Δt_(F) forms the sensor signal.

[0073] In analogy to further above, however, also the sensor signalt_(F) and the disturbance values from the (three-layer- or four-layer-)mode equations for the two polarizations and for several wavelengths andmode numbers can be determined, this under the prerequisite, that thesystem of equations can be numerically solved.

[0074] Fundamentally also the layer thicknesses are to a certain degreedependent on the temperature (also refer to Applied Optics 20 (1981),2280-2283), which fact, however, has been neglected in the formulas (1),(2) and (3) in the approximation. It is, however, also possible withrespect to at least one specimen select such a combination of (several)layers and substrate, that the temperature coefficient of the waveguideor of the grating coupler (resp., of the waveguide grating structure)with respect to at least one sensor signal S (=α(TE), α(TM),α(TM)−α(TE), N(TE), N(TM), N(TM)−N(TE), t_(F), t_(A) etc.) ispractically zero (the sensitivity, however, still remains high). Inthis, e.g., one of the layers involved can be an SiO₂ layer.

[0075] The absolute temperature coefficient with respect to a sensorsignal S is dS/dT, the corresponding relative temperature coefficient is(1/S)(dS/dT). with dN(TE)/dT=0 or dN(TM)/dT=0, also S=N(TE) or S=N(TM)become interesting sensor signals independent of the temperature.

[0076] If in a “sensing pad” only one grating is present, then, forexample, with the reflection arrangement described in the Europeanpatent application 0 482 377 A2 (possibly with a sensor chip withtilting of the grating plane versus the lower substrate plane)measurements are possible. The measurement with the reflectionarrangement on weakly or more strongly modulated (mono-diffractive ormulti-diffractive) waveguide gratings (homogeneous gratings,superimposed gratings with different grating periods and/or gratingorientations, chirped gratings, etc.) can be carried out both for TE- aswell as for TM-waves. The mode excitation can take place from the left,the readout from the right or vice-versa. For example, in a “sensingpad” the excitation of the TE-wave can take place from the left and theexcitation of the TM-wave from the right. In a second “sensing pad”(possibly with a different grating period and/or grating orientation)the excitation can be in reverse to the first “sensing pad”. The readoutcan take place in the reflected and transmitted light field in thezero-th or higher diffraction order. With this, it is also possible todetermine absolute coupling angles with the reflection arrangement. Themeasured absolute coupling angle, which in essence in case of the samegrating period (and the same diffraction order) corresponds to half ofthe angle difference between the two corresponding resonance minimums,does not change, even if the sensor chip is slightly tilted. From theabsolute coupling angles for the TE-wave or the TM-wave, thecorresponding effective refractive indexes and further integratedoptical measured values can be determined, such as, e.g., the layerthickness d_(F) of the waveguiding film in the three-layer waveguidemode., etc.

[0077] It is of course also possible that in the first “sensing pad” theexcitation of the TE- and TM-wave takes place from the left and thereadout from the right (if so required with only one CCD) and in thesecond “sensing pad” (possibly with a differing grating period) inreverse.

[0078] It is also possible that the plane of incidence of the beamguidance responsible for the reference path (second “sensing pad”) isrotated or tilted or rotated and tilted versus the plane of incidence ofthe beam guidance responsible for the signal path (first “sensing pad”).First and second “sensing pads” can also coincide. The coupling anglesfor the TE-wave and the TM-wave can also be measured at differentwavelengths and/or mode numbers.

[0079] A further “sensing pad” arrangement consists of two (identical)chirped gratings (gratings with graduated grating period), whereby onegrating serves as in-coupling grating and one grating as out-couplinggrating. Chirped gratings are known from the literature (refer to, e.g.,the patent application WO 97/09594). The signal “sensing pad” and thereference “sensing-pad” can have the same or also an opposing chirpdirection (direction, in which the grating period changes (e.g., becomesbigger), it is vertical to the direction of the mode propagation). The“sensing pads” can be coated with the same (bio-)chemo-sensitive layer(see below) or else have differing ((biolayers, whereby the((bio-)chemo-sensitive) layer of the second “sensing pad” (reference“sensing pad”) is a different (bio-)chemo-sensitive layer or anonspecific ((bio-)chemo-sensitive) layer with or without “non-specificbinding” (e.g., dextran without identification molecule) or else apurely protective layer.

[0080] The chirped grating, which is responsible for the in-coupling, isilluminated with a (not wedge-shaped or if so applicable (slightly)wedge-shaped) light strip, of which a certain proportion, in the case ofwhich the in-coupling conditions are fulfilled, is in-coupled to thewaveguide. It is also possible to simultaneously illuminate thein-coupling chirped gratings of the signal-“sensing pad” andreference-“sensing pad” (if so required with a single, longer lightstrip) (if so required with 45° linear polarized or circular (orelliptically) polarized light (for the excitation of modes of bothpolarizations)). In the case of a excitation of modes of differingpolarization under a fixed angle of incidence, the corresponding gratingperiods of the two “sensing pads” are different.

[0081] In the case of the same polarization and opposite chirp directionof signal “sensing pad” and reference “sensing pad” (both “sensing pads”are coated with the same (biolayer), the two out-coupled light spots onthe basis of the (biobinding travel (practically) vertical to thedirection of propagation of the modes towards one another or away formone another (depending on the chirp direction or the chirp orientation).The position of the light spots is measured with PSDs or with a one- (ortwo-) dimensional CCD. Through the change of the distance of the twolight spots, the change of the effective refractive index of thecorresponding polarization can be calculated. The distance of the twolight spots is an absolute value, because the distance of the two lightspots is independent of displacements or of small tilting. (If thesignal and reference paths have a differing polarization, then themeasuring signal ΔN(TM)−ΔN(TE) can be determined.) In the case of thesame polarizations, same chirp directions of the two “sensing pads”, butdiffering (bio-)chemo-sensitive layer (e.g., on the signal path aspecific layer, on the reference path a non-specific layer), thedistance of the two light spots forms a referenced (absolute) sensorsignal.

[0082] The arrangement described in the paragraph before the last onecan once again be duplicated for the other polarization (with anadaptation of the grating period) and can once again as a whole beconsidered as reference path for the complete layout described in theparagraph before the last one (to be interpreted now as signal path).Here the change of the effective refractive index of the otherpolarization can be measured. (If, however the polarization(s) remains(remain) the same and instead another chemo-sensitive layer (with orwithout non-specific binding) or a non-specific layer with or withoutnon-specific binding (e.g., dextran layer without identificationmolecule) is utilized, then the referenced measured values (measuredvalue (first arrangement) measured value (second arrangement)) of themeasured values ΔN (TE) or ΔN(TM) or ΔN(TM)−ΔN(TE) can be determined).

[0083] It is also possible, that only the in-coupling grating is presentas a chirped grating, the out-coupling grating, however, e.g., ismono-diffractive (or multi-diffractive). The two light spots describedthree paragraphs above do not anymore travel vertically to the modepropagation direction on the basis of the (bio-)chemical binding,because the out-coupling grating also deflects in the plane of incidence(possibly better referred to as the outlet plane) (i.e., theout-coupling angle changes).

[0084] If on the other hand for signal “sensing pad” and reference“sensing pad” one operates with three gratings each (one in-couplinggrating and two out-coupling gratings or two in-coupling gratings andone out-coupling grating), whereby modes in forward and reversedirections are excited, if the in-coupling grating is a chirped grating(with the same or with opposite chirp orientation between the two“sensing pads”) and if the in-coupling gratings are, e.g.,mono-diffractive (or multi diffractive), then displacements or tiltingof the x- and y-axis (orientation of the axis as in FIG. 1) can beidentified and eliminated on the basis of the absolute measurement. Forthe chemo-sensitive layer(s), the same remarks are applicable as in thecase of the “sensing pad”, which consists of two chirped gratings. If,for example, only the (absolute) out-coupling angle (and/or measuredvalues which can be derived from it) are considered as sensor signals,then the chirped in-coupling grating can also be illuminated with alight strip impinging in a wedge-shape. Here too, the above arrangement(signal and reference path) can be duplicated and considered as a newreference path (with or without differing chemo-sensitive layer or witha non-specific layer) to the above arrangement (now as a whole as signalpath).

[0085] The specimen liquid(s) is (are) brought into contact with thewaveguide or with the chemo-sensitive substance(s) through a “well” or amatrix of “wells”, a through-flow cell or a matrix of through-flowcells, a capillary vessel or a matrix of capillary vessels.

[0086] Two-dimensional arrangements lead, e.g., to a “microplate” with96, 384, 1536 “wells”, etc. (But also other two-dimensional formats(e.g., disks) are possible). But also the manufacturing of individualstrips (one-dimensional arrangement) is possible.

[0087] The strips can, e.g., also be inserted into the frame of amicroplate. The “wells” can be affixed to the sensorchip plate, whichcontains the waveguide grating structure units as separate specimen cell(resp., specimen cell plate). It is however, also possible to providethe substrate itself with indentations in such a manner, that theseindentations already assume the function of the “wells” or of thethrough-flow vessels or of the capillary vessels. In the two lattercases, the sensorchip plate has to be covered with a covering plateequipped with bores. In the first case, the sensorchip plate can becovered with a covering plate (without bores), in order to, e.g.,prevent evaporation. The bores serve for the supply or carrying away ofthe specimen or for the ventilation. In preference, one works withplastic material and injection-moulding techniques or hot-embossingtechniques. But also sol-gel techniques, UV-hardening techniques fororganic/inorganic composites, glass embossing techniques (hot-embossingor (injection-)moulding), etching techniques, material removal by laser,etc., present an alternative.

[0088] The grating structures can be manufactured using embossingtechniques (hot-embossing, cold-embossing, UV-hardening) orinjection-moulding techniques out of plastic material, sol-gel, glass,UV-hardening organic or inorganic materials or organic/inorganiccomposites, ormoceres or nanomeres, with removal by laser in conjunctionwith interferometry, holography and/or phase mask techniques,photolithography in conjunction with wet or dry etching, using photopolymerization (refer to, e.g., P. Coudray et al., Crit. Rev. Opt. Sci.Tech. (SPIE) CR68 (1997), 286-303) or using casting techniques (e.g., insol-gel), etc. Plastic injection-moulding techniques, such as areutilized in the manufacturing of compact discs (e.g., out ofpolycarbonate), are particularly suitable. The grating structure can bein (resp., on) the substrate or in (resp., on) a layer or can be presentin a combination of these. The grating structures can be surface reliefgratings (resp., interface relief gratings) or refractive-index gratings(resp., volume gratings) or combinations of these. The waveguiding filmcan be a sol-gel layer (SiO₂—TiO₂, TiO₂, (non-porous) high-refractionlead-silicate glass, etc)., an organic/inorganic composite layer, apolymer layer, a PVD-, a CVD-, a PE-CVD-, a PI-CVD layer, aphoto-polymerizable, high-refraction layer (e.g., photo-polymerizableTiO₂), etc. or combinations of these. It is also possible that a layer(with in preference low refractive index, made, e.g., of SiO₂ sol-gel,SiO₂—TiO₂ sol-gel (with low TiO₂ content), lead-silicate glass, sol-gel(with low lead content), sol-gel similar to float-glass, etc.) containsthe grating structure and a second layer (e.g., made of SiO₂—TiO₂, TiO₂,Ta₂O₅, HfO₂, ZrO₂, Nb₂O₅, Si₃N₄, lead-silicate glass, etc.) forms thewaveguiding film. Due to the fact that the softening point oflead-silicate glass is significantly below the softening point of glass,the lead-silicate glass can be made to melt without the glass in doingso. The melting process leads to a practically pore-free lead-glass. Ifthe substrate and the layer(s) have similar thermal expansioncoefficients, then the formation of micro-fissures can be prevented(micro-fissures increase the dampening values of the modes). PVD- andCVD-processes make possible the manufacture of very compact waveguidingfilms.

[0089] The substrate can be made of a plastic material (e.g.,polycarbonate, PMMA, polystyrene, etc.), sol-gel or of glass(float-glass, specimen slides, soda-lime glass, boro-silicate glass,alkali-free glass, quartz, etc,). However, also the grating material canbe utilized as substrate material (e.g., ormocere, UV-hardeningmaterial).

[0090] The specimen cell can consist of indentations (holes, bores),also, however, of through-flow cells. These through-flow cells can alsobe configured in such a manner, that the specimen liquid can be suppliedusing the pin of a pipette robot. It is also possible, that a secondspecimen cell consisting of through-flow vessels is inserted into afirst specimen cell with indentations (holes).

[0091] In the substrate or anywhere on the sensorchip plate, positioningmarks (holes, indentations, pins, elevations, etc.) can be inserted oraffixed. These positioning marks guarantee that the impinging light beamhits the in-coupling grating(s). The identification of the positioningmarks is effected through the measuring unit.

[0092] For the separation of the light wave carrying the sensor signalfrom other light waves (e.g., from the light wave reflected from thebottom of the substrate), it is also advantageous if the waveguidingfilm is not plane-parallel to the bottom of the substrate. Thisnon-plane-parallelism can be present, among others, in the form ofwedges, prisms, cylinder prisms, spherical lenses, cylinder lenses, etc.

[0093] With the chemo-sensitive waveguide structure, not only a directdetection, but also marking detections can be carried out. Theutilization of a “refractive index label” (e.g., of a little plasticball (e.g., a small latex ball), bio-chemical and biological fragments,etc.), can already be considered as a marking detection. With it, forexample, sandwich assays or competition assays can be implemented.

[0094] As light source a monochromatic light source, such as, e.g., a(pulsed) laser, a (pulsed) laser-diode, a (pulsed) LED with or withoutfilter in the (infra)red or blue-green or ulta-violet spectral range, isadvantageously used. But also thermal light sources with a filter can beutilized. Red-blue-green or ultra-violet wavelengths have the advantagesthat (a) the sensitivity for the direct detection increases and (b) withthe same light source apart from “direct sensing” also fluorescence-,phosphorescence or luminescence tests can be conducted, whereby theexcitation of the fluorescence, phosphorescence or luminescence takesplace through the evanescent wave (in the form of a TE-wave or TM-waveor TE- and TM-wave). The fluorescence-, phosphorescence- orluminescence-light can be observed as a plane wave or also as a guidedwave. The fluorescent light caused by the TE-exciting wave (resp., itsintensity) can be referenced or compared with the fluorescent light(resp., its intensity) caused by the TM-exciting wave, wherebyadvantageously the intensity of the exciting wave is jointly taken intoaccount and possibly the detectors are made sensitive to polarization bythe utilization of polarizers. The guided fluorescence-,phosphorescence- or luminescence-light wave can be out-coupled through agrating and conveyed to a detector. Sandwich-assays, competitionassays,etc., can be carried out, whereby at least one participating bindingpartner is fluorescence(phosphorescence-, luminescence-) marked. The(bio-) chemosensitive layer can be present on the grating, or can also,however, be present solely between the gratings or outside the grating.

[0095] Fluorescence, phosphorescence or luminescence measurementsmanifest a low dependence on temperature. For fluorescence,phosphorescence or luminescence measurements, purely inorganic waveguidegrating structures are particularly suitable (the grating ismanufactured in glass or sol-gel (e.g., SiO₂) or in the inorganicwaveguiding film, waveguiding film made of inorganic material (e.g.,Si₃N₄ or oxide layers such as, e.g., TiO₂ or Ta₂O₅ or lead-silicatelayers, etc.)). Inorganic materials manifest, e.g., a low fluorescenceof their own. If one works with a plastic material substrate, then it isrecommended to apply an inorganic low-refraction intermediate layer(e.g., SiO₂) to the plastic substrate. The layer thickness of thisintermediate layer has to be selected as great enough, so that theevanescent wave running in it practically does not anymore “see” theplastic substrate. As a result of this, at least the proprietaryfluorescence generated by the guided light wave is strongly reduced.

[0096] If both the intensity of the (possible out-coupled) excitationwave as well as that of the (possibly out-coupled) emission wave aremeasured, then various interference factors (such as, e.g., those, whichare caused by intensity fluctuations of the exciting wave) can beeliminated by referencing. The referenced sensor signal is then, forexample, the intensity of the fluorescence (phosphorescence,luminescence) divided by the intensity of the exciting light wave. Theintensity of the exciting wave can be measured prior to the penetrationof the exciting wave into the chemo-sensitive layer or after the exit ofthe exciting wave from the chemo-sensitive layer or at both points. Withreferenced marking detections, absolute kinetic measurements as well asabsolute end point measurements can be carried out. In the case ofabsolute (end point) measurements, the (bio-)chemical interaction on thewaveguide grating structure can also take place outside the measuringinstrument.

[0097] It is also to be noted that with a (digital or analogue) PSD(position sensitive detector) (one-dimensional or two-dimensional), aphoto-diode-array (PDA) (one-dimensional or two-dimensional) or aCCD-array (one-dimensional or two-dimensional), not only (positions of)light field distributions but also intensities can be measured.

[0098] It is possible (but it is not absolutely necessary) to in partwith the same detectors carry out a direct detection as well as amarking detection (on a fluorescence, phosphorescence or luminescencebasis). Evanescence field excitation can also be combined with measuringtechniques resolved over time (e.g., time-resolved fluorescence orluminescence). In the case of time-resolved measuring techniques, themarking is excited with a pulsed light wave (primarily in the visible orultra-violet wavelength range). The fading times for the fluorescence(luminescence, phosphorescence) of free and bound marking molecules(with or without energy transfer between two different fluorescencemolecules (refer to, e.g., the F ö rster theory in CIS BioInternational, November 1995, No. 3)) are different. The depth ofpenetration of the guided excitation wave into the specimen determinesthe “observation volume”.

[0099] It is interesting that with the waveguide grating structurespresented here absolute measurements can be carried out both free ofmarking as well as fluorescence (phosphorescence, luminescence) marked(if so required also simultaneously). In both cases, for the in-couplingof light one can make do without movable mechanics. It goes withoutsaying that also (continuous) kinetic measurements or real-timemeasurements can be carried out on a non-absolute basis.

[0100] With a “sensing pad” (signal path) (with or withoutchemo-sensitive layer), it is also possible to carry out lightabsorption measurements, inasmuch as the intensities of the light beamsout-coupled (through grating, prism, taper or front side) (if sorequired with the same detectors) can be measured, possibly also atdifferent wavelengths. The change in light absorption can come aboutdirectly or indirectly (e.g., through enzymes) through the(bio-)chemical interaction of the specimen with the chemo-sensitivelayer or through the specimen itself or through the reactions takingplace in the specimen (with or without an additional reaction partner).The chemo-sensitive layer can be present on the grating, between thegratings or outside the grating. Fluctuations in light intensity of thelight source can be eliminated by referencing (e.g., with abeam-splitter and a reference detector or through a not in-coupleddiffraction order and a reference detector). Referencing can also becarried out by the method in which the second “sensing pad” (referencepath) is covered with a protective layer and therefore cannot interactwith the specimen. The referenced sensor signal is then the intensity ofthe signal path detector divided by the intensity of the reference pathdetectors.

[0101] The protective layer, however, can also be a (porous)(chemo-sensitive) layer, which has no specificity (with or without“non-specific binding”) or specificity for another ligand.

[0102] Referencing can also (but does not have to) take place throughone half of a “sensing pad” (e.g., signal path: mode in forwarddirection, reference path: mode in reverse direction).

1. An optical sensor for the characterization or the detection or thedetection and characterization of a chemical or bio-chemical substancecomprising at least one optical waveguide with a substrate, awaveguiding material, a cover medium and at least one waveguide gratingstructure, at least two sensing pads comprising at least oneunidiffractive or multidiffractive grating, at least one of the sensingpads acting as sensor pad and comprising a sensor chemosensitive orbio-chemosensitve substance, and at least one of the sensing pads actingas reference pad and comprising a reference chemosensitive orbio-chemosensitive substance, light source means for the simultaneousillumination of gratings of the sensor pad and of the reference pad;detection means for detection of positions or of intensities or ofpositions and of intensities of at least two light distributionproportions, which, on the detection means, are not superimposed on oneanother and which are emitted or coupled out or emitted and coupled outfrom the waveguide grating structure into the substrate or into thecover medium or into the substrate and into the cover medium; means forthe generation of a referenced sensor signal through the evaluation ofthe detected light distribution, of the detected positions or ofintensities of the at least two light distribution proportions or of acombination of these.
 2. The optical sensor according to claim 1,wherein the sensor pad and the reference pad each comprise at least onein-coupling grating and at least one out-coupling grating.
 3. Theoptical sensor according to claim 1, wherein a lens system is arrangedbetween the waveguide and the detection means.
 4. The optical sensoraccording to claim 1, comprising one detector for the detection of theat least two light proportions, said detector comprising means forresolving the positions or the intensities or the positions and theintensities of the at least two light proportions.
 5. The optical sensoraccording to claim 1, wherein the detector is a 1-dimensional or a2-dimensional photodiode array or CCD camera or a 1-dimensional or2-dimensional analogue position sensitive detector or a 1-dimensional or2-dimensional digital position sensitive detector.
 6. The optical sensoraccording to claim 1 wherein a waveguide grating structure comprisesmeans for producing at least four emitted or out-coupled or emitted andout-coupled light fields corresponding to the forward and the rearwarddirection of the transverse electric mode and the transverse magneticmode.
 7. The optical sensor according to claim 1 comprising means forconducting at least two of a direct sensing test and of a fluorescencetest and of a luminescence test and of a phosphorescence test using onlyone light source.
 8. The optical sensor according to claim 1, whereinthe two sensing pads are adjacent to one another.
 9. The optical sensoraccording to claim 1 wherein the two sensing pads are located in oneanother or one above the other.
 10. The optical sensor according toclaim 1 wherein the grating or gratings of the sensor pad have the samegrating period or grating periods as the grating or gratings of thereference pad.
 11. The optical sensor according to claim 1 whereinsensor pad and reference pad differ from each other in the gratingperiod of at least one grating.
 12. The optical sensor according toclaim 1 wherein at least one grating is situated in a volume or on abordering surface or in a volume and on a bordering surface of amaterial contained in the waveguide.
 13. The optical sensor according toclaim 2, wherein one sensing pad of the waveguide grating structure unitcontains two out-coupling gratings and an in-coupling grating situatedbetween the out-coupling gratings, or two in-coupling gratings and anout-coupling grating situated between the in-coupling gratings.
 14. Theoptical sensor according to claim 1, wherein a “well” or a matrix of“wells” is affixed onto the waveguide grating structure or is insertedinto the waveguide grating structure.
 15. The optical sensor accordingto claim 1, wherein a flow-through cell or a matrix of flow throughcells or a capillary vessel or a matrix of capillary vessels are affixedonto the waveguide grating structure or is inserted into the waveguidegrating structure.
 16. The optical sensor according to claim 1, whereinat least the reference chemosensitive or bio-chemosensitive layer showsessentially no nonspecific binding.
 17. The optical sensor according toclaim 1, wherein the two chemosensitive or bio-chemosensitive layersassigned to the two sensing pads manifest a differing specifity.
 18. Theoptical sensor according to claim 1, wherein at least the referencechemosensitive or bio-chemosensitive substance shows essentially nononspecific binding and no specificity.
 19. The optical sensor accordingto claim 1, wherein the sensor pad and the reference pad are arranged atleast partially at a distance from each other.
 20. The optical sensoraccording to claim 1, wherein said chemosensitive or bio-chemosensitivesubstances comprise dextran, and wherein the specificities of the saidchemosensitive or bio-chemosensitive substances of the sensor pad and ofthe reference pad are different.
 21. The optical sensor according toclaim 1, wherein at least one of said chemosensitive orbio-chemosensitive substances is dextran essentially without anyidentification molecules.
 22. The optical sensor according to claim 1comprising at least one capillary vessel in which a specimen may bebrought into contact with the waveguide or with the chemosensitive orbio-chemosensitive substance or the chemosensitive or bio-chemosensitivesubstances or with both, with the waveguide and with the chemosensitiveor bio-chemosensitive substance or the chemosensitive orbio-chemosensitive substances.
 23. The optical sensor according to claim1, wherein said light source means comprises a laser diode.
 24. Theoptical sensor according to claim 2, wherein said in-coupling gratingsof said sensor pad and of said reference pad are arranged in a mannerthat illumination of both in-coupling gratings by one single light beamis enabled.
 25. The optical sensor according to claim 24, wherein thegrating defines a plane with a first direction (y) parallel to thegrating lines and with a second direction (x) perpendicular to saidfirst direction (y), wherein said gratings of said sensor pad and ofsaid reference pad or said incoupling gratings of said sensor pad and ofsaid reference pad lie in immediate vicinity to each other and arespaced in said first direction (y).
 26. The optical sensor according toclaim 1, wherein said sensor pad and said reference pad togethercomprise one chemosensitive or bio-chemosensitive substance, to whichtwo different specimen liquids can be brought into contact.
 27. Theoptical sensor according to claim 1, wherein said at least onewaveguideing material comprises said bio-chemosensitive orchemosensitive substance or said bio-chemosensitive or chemosensitivesubstances.
 28. The optical sensor according to claim 1 comprising meansfor the immovable fixation of the waveguide grating structure relativeto the light source means and the means of detection for the purpose ofcarrying out a measurement.
 29. The optical sensor according to claim 1,wherein the substrate is made of plastic material.
 30. The opticalsensor according to claim 1, wherein the substrate is coated by anintermediate layer of low refractive index.
 31. The optical sensoraccording to claim 1, wherein the waveguiding material is a waveguidingfilm comprising at least one layer.
 32. The optical sensor according toclaim 31, wherein the waveguiding film comprises at least one layer ofhigh refractive index and one polymer layer.
 33. The optical sensoraccording to claim 1, wherein two light distribution proportions showdifferent wavelengths and different polarizations.
 34. The opticalsensor according to claim 1, further comprising a reflectionarrangement.
 35. A sensor chip for the characterization or the detectionor for the detection and characterization of a chemical or bio-chemicalsubstance, comprising at least one optical waveguide with a substrate, awaveguiding film, and at least one waveguide grating structure, thesubstrate comprising a bottom, and said waveguide grating structurebeing configured such as to form at least two sensing pads, eachcomprising a chemosensitive or bio-chemosensitive substance, saidwaveguiding film not being plane-parallel to the bottom of thesubstrate.
 36. The sensor chip according to claim 35, wherein the bottomof the substrate comprises at least one of wedges, prisms, cylinderprisms, spherical lenses, and cylinder lenses.
 37. The sensor chipaccording to claim 35, wherein the substrate is made of a plasticmaterial.
 38. The sensor chip according to claim 35, wherein thesubstrate is provided with an intermediate layer of low refractiveindex.
 39. The sensor chip according to claim 35, wherein thewaveguiding film comprises at least one layer.
 40. The sensor chipaccording to claim 39, wherein the waveguiding film comprises at leastone layer of a high refractive index and one polymer layer.
 41. Thesensor chip according to claim 35, wherein at least one gratingstructure comprises a UV hardening organic or inorganic material or anUV hardening organic/inorganic composite.
 42. The sensor chip accordingto claim 35, wherein said chemosensitive or bio-chemosensitivesubstances comprise dextran, and wherein the specificities of the saidchemosensitive or bio-chemosensitive substances of the sensor pad and ofthe reference pad are different.
 43. The sensor chip according to claim35, wherein at least one of said chemosensitive or bio-chemosensitivesubstances is dextran essentially without any identification molecules.44. A sensor chip for the characterization or detection or for thedetection and characterization of a chemical or bio-chemical substance,comprising at least one optical waveguide with a substrate comprising abottom, a waveguiding film, and at least one waveguide gratingstructure, and said waveguide grating structure forming at least twosensing pads, each comprising a chemosensitive or biochemosensitivesubstance, each sensing pad comprising at least one in-coupling gratingand at least one out-coupling grating, the in-coupling gratings of thetwo sensing pads being arranged next to each other.
 45. The sensor chipaccording to claim 44, wherein the grating defines a plane with a firstdirection (y) parallel to the grating lines and with a second direction(x) perpendicular to said first direction (y), wherein said gratings ofsaid sensor pad and of said reference pad or said incoupling gratings ofsaid sensor pad and of said reference pad lie in immediate vicinity toeach other and are spaced in said first direction (y).
 46. The sensorchip according to claim 44, wherein the substrate is made of a plasticmaterial.
 47. The sensor chip according to claim 44, wherein thesubstrate is provided with an intermediate layer of low refractiveindex.
 48. The sensor chip according to claim 44, wherein thewaveguiding film comprises at least one layer.
 49. The sensor chipaccording to claim 48, wherein the waveguiding film comprises at leastone layer of a high refractive index and one polymer layer.
 50. Thesensor chip according to claim 44, wherein said chemosensitive orbio-chemosensitive substances comprise dextran, and, wherein thespecificities of the said chemosensitive or bio-chemosensitivesubstances of the sensor pad and of the reference pad are different. 51.The sensor chip according to claim 44, wherein at least one of saidchemosensitive or bio-chemosensitive substances is dextran essentiallywithout any identification molecules.
 52. The sensor chip according toclaim 44, wherein at least one grating structure comprises a UVhardening organic or inorganic material or an UV hardeningorganic/inorganic composite.
 53. A sensor chip for the characterizationor detection or for the detection and characerization of a chemical orbio-chemical substance, comprising at least one optical waveguide with asubstrate comprising a bottom, a waveguiding film, and at least onewaveguide grating structure, the sensor chip comprising an array ofcapillary flow cells or an array of capillary vessels.
 54. A sensor chipfor the characterization or detection or the detection andcharacterization of a chemical or bio-chemical substance, comprising atleast one optical waveguide with a substrate, a waveguiding film, and atleast one waveguide grating structure, the substrate comprising abottom, a waveguiding film, and at least one waveguide gratingstructure, and said waveguide grating structure being configured so asto form at least two sensing pads, each comprising a chemosensitive orbio-chemosensitive substance, each sensing pad comprising oneunidiffractive or multidiffractive grating for use in reflection typemeasurements.
 55. The sensor chip according to claim 54, wherein thegrating defines a plane with a first direction (y) parallel to thegrating lines and with a second direction (x) perpendicular to saidfirst direction (y), wherein said gratings of said sensor pad and ofsaid reference pad or said incoupling gratings of said sensor pad and ofsaid reference pad lie in immediate vicinity to each other and arespaced in said first direction (y).
 56. The sensor chip according toclaim 54, wherein the substrate is made of a plastic material.
 57. Thesensor chip according to claim 54, wherein the substrate is providedwith an intermediate layer of low refractive index.
 58. The sensor chipaccording to claim 54, wherein the waveguiding film comprises at leastone layer.
 59. The sensor chip according to claim 54, wherein thewaveguiding film comprises at least one layer of a high refractive indexand one polymer layer.
 60. The sensor chip according to claim 54,wherein said chemosensitive or bio-chemosensitive substances comprisedextran, and wherein the specificities of the said chemosensitive orbio-chemosensitive substances of the sensor pad and of the reference padare different.
 61. The sensor chip according to claim 54, wherein atleast one of said chemosensitive or bio-chemosensitive substances isdextran essentially without any identification molecules.
 62. The sensorchip according to claim 54, wherein at least one grating structurecomprises a UV hardening organic or inorganic material or an UVhardening organic/inorganic composite.
 63. An optical process for thecharacterization or for the detection or the detection andcharacterization of a chemical or bio-chemical substance in a specimenby means of a waveguide grating structure containing at least onewaveguide grating structure unit, wherein the specimen is brought intocontact with the waveguide structure in at least one contact zonecomprising a sensor chemosensitive or bio-chemosensitive substance and areference chemosensitive or bio-chemosensitive substance, in thewaveguide structure in the region of the at least one contact zone,simultaneously exciting at least two light waves through the waveguidegrating structure unit or at least one grating of the sensor pad and onegrating of the reference pad of the waveguide grating structure unit areilluminated simultaneously, and bringing at least one light wave intointeraction with the specimen, wherein the light waves differ in atleast one of their polarization, their mode number, their wavelength andof their position on the waveguide grating structure, or the sensorchemosensitive or bio-chemosensitive substance and the referencechemosensitive or bio-chemosensitive substance are different, or whereat least one light wave is brought into interaction with a firstspecimen and a second light wave is brought into interaction with asecond specimen; detecting light in at least two differing proportions,which are not superimposed on the detection means and of which at leastone proportion originates from the at least one contact zone, generatingat least one referenced measured signal by the evaluation of thedetected light.
 64. The process according to claim 63, wherein themeasured signal is generated on the basis of a direct detection.
 65. Theprocess according to claim 63, wherein the measured signal is a layerthickness or a change in layer thickness according to the solution ofthe mode equation for at least one polarization, at least one wavelengthand at least one mode number.
 66. The process according to claim 63,wherein the measured signal is a layer thickness or a change in layerthickness according to the solution of a linear system of equations forat least one polarization, at least one wavelength and at least one modenumber.
 67. The process according to claim 63, wherein the measuredsignal is generated on the basis of a marking detection.
 68. The processaccording to the claim 63, wherein both a measured signal belonging tothe direct detection as well as a measured signal belonging to themarking detection are generated.
 69. The process according to claim 63,wherein the measured signal is resolved as a function of time.
 70. Theprocess according to claim 63, wherein a waveguide structure or awaveguide grating structure is selected in such a manner, that thetemperature coefficient of the waveguide structure or of the waveguidegrating structure is practically zero with respect to at least onespecimen and with respect to at least one measured signal.